A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.
Ranges are often expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment.
Where methods or processes are expressed herein as lists or sequences of steps, including as lists of numbered, lettered, or bulleted steps, the order in which these steps is presented is not intended to imply any particular order or timing of the steps in the method, unless a particular order is required or is explicitly stated. Unless otherwise provided, therefore, the steps of each disclosed method can be carried out in any sensible order, as is clear to one skilled in the art i.e. any order which is suitable to achieve the stated purpose or product of that method. Also, unless expressly stated, the presence of additional steps in the methods is not excluded.
The novel thymidilate synthase inhibitor (2,R)-((4,S)-carboxy-4-(4,N-(((6S)-2-(hydroxymethyl)-4-oxo-3,4,7,8-tetrahydro-3H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino)benzamido)butanamido)pentanedioic acid 1, as its trisodium salt 2 (also known as CB300945, BCG945, or ONX-0801) is a very potent inhibitor of thymidilate synthase, with an IC50 of 3 nM for the enzyme.
Unlike most thymidilate synthase inhibitors such as Methotrexate, and Pemetrexed, 2 does not require polyglutamoylation for its activation, or for cellular retention, and it is not a substrate for the polyglutamoylation synthase enzyme.
Furthermore, 2 is not a substrate for the commonly expressed reduced folate carrier protein (RFCP) with a Ki>250 μM, and does not pass readily through regular cell membranes. However, it does bind to the folate receptor α (FRα) with a very high, subnanomolar affinity (Ki 0.5 nM), and can be transported into cells at a modest rate via that receptor, even when serum levels of 2 are very low.
Once in the cells, 2 inhibits thymidilate synthase and, as it is degraded and expelled from cells slowly, it produces a long lasting inhibition of thymidilate synthase. This results in DNA processing errors, which initiate a repair cycle, which is ultimately futile due to the lack of thymidine triphosphate for incorporation into the DNA. This leads to apoptotic DNA damaging responses and cell death.
This is the general mode of action of thymidilate synthase inhibitors, and clinical experience shows that they have a strong cytotoxic effect in human tumors, but their use is hampered by their rather broad spectrum toxicity, where target tissues, especially bone marrow, are seriously affected by the cytotoxicity of the drugs.
The current generation of thymidilate synthase inhibitors, being good substrates for the ubiquitously expressed RFCP, are distributed well into most tissues, whereas 2 is only distributed efficiently into tissues which express, or preferably overexpress, FRα. Relatively few tissues endogenously express FRα, even at modest levels. Most tissues that do express it do so in a polarized fashion on the distal face of cells, meaning that there are no transporters on the side of the cell facing the circulatory endothelium. This has the consequence that even in these tissues, penetration of 2 tends to be very inefficient. This means that high circulating levels of 2 in the plasma lead to rather low systemic toxicities.
If the same were true of tumors, it would also be expected to lead to low anti-tumor efficacy. However, there are certain classes of tumor tissues which tend to overexpress FRα. Being tumor cells, they have lost their polarity and express FRα on both apical and distal faces. Thus, these tumor tissues have the unusual propensity of being able to concentrate quite large, cytotoxic, doses of 2 in their cells from plasma drug levels low enough to have very little toxic effect. This leads to an enhanced therapeutic index for 2 in the overexpressing tumor types.
There are several tumor types which overexpress FRα, the most notable of which is ovarian cancer, where 90% of the commonest tumor type, the mucinous form of the cancer, overexpress FRα. Thus 2 is a very attractive chemotherapeutic agent for the treatment of FRα-overexpressing cancers, especially ovarian cancer, although it is not limited to that one tumor type, with uterine cancer, mesothelioma and kidney cancer, amongst others, having high percentages of FRα overexpression.
Compounds 1 and 2, along with several close congeners, are disclosed in WO2003/020748, albeit as a 1:1 mixture of diastereoisomers at the 6-position.

WO 2003/020748 also reveals how to synthesize compound 1, once again as a mixture of diastereoisomers at the 6-position.
As shown in Scheme 1, the cyclopentaquinazolinylaminobenzoic acid 3 is condensed with tri-O-t-butyl L-glutamyl-γ-D-glutamate, 4, using diethylphosphononitrile and triethylamine in DMF at room temperature to give tri-O-t-butyl N-{N-{4-[N-((6RS)-2-(2,2-dimethylpropionyloxymethyl)-4-oxo-3,4,7,8-tetrahydro-3H-cyclopenta[g]quinazolin-6-yl)-N-(prop-2-ynyl)amino]benzoyl}-L-γ-glutamyl}-D-glutamate 5, in 62% yield, on a low milligram scale. The three t-butyl esters are then removed by treatment with TEA at room temperature for 1 hour, the solvent is stripped at room temperature or below, and a solvent exchange is carried out to 1:1 methanol:water. The pH is raised to 12 with sodium hydroxide solution, and the pivaloyl ester hydrolyzed at room temperature. The solution is then acidified to pH 4 with 1M hydrochloric acid and precipitated at 0° C. to give acid 1 (6RS mixture) in 47% yield after filtration and drying.

In U.S. Pat. No. 7,250,511 the same synthesis is disclosed for Compound 1, but a more useful variant is revealed for compound 6, as shown in Scheme 2. Compound 6 is simply compound 1, N-methylated on the D-Glu amine. In this case, depivaloylated core acid 7 is coupled with 8, the N-methylated analogue of dipeptide tri-ester 4, under the same conditions as described for the coupling of 3 and 4, to give the protected compound 9 in 40% yield. The three t-butyl esters are then removed by treatment with TEA at room temperature for 1 hour, and the solvent was then stripped at room temperature or below, and the residue was taken up in water, basified to pH 10 with dilute sodium hydroxide, and the final acid was precipitated with 1M hydrochloric acid, filtered and dried as previously to give acid 6 (6RS mixture) in 77% yield.
A method of resolving compounds of general formula (I) is revealed in WO 94/11354, in order to get the more active, and hence more desired, (6S)-enantiomers. This method involves taking racemic acids of formula (I) and condensing them with a chiral amino acid, preferably L-glutamic acid, or (S)-2-aminoadipic acid to form an amide of formula (II) as a 1:1 mixture of diastereoisomers.

Use of an appropriate protease, such as Carboxypeptidase G2 selectively hydrolyzes the 6R-diastereoisomer, allowing for a straightforward separation of 6S-(II) from 6R-(1). The 6S-(II) is then hydrolyzed enzymatically to 6S-(I) in >98% enantiomeric excess. It is assumed that this process was carried out on acid 3, or possibly acid 7, since U.S. Pat. No. 7,528,141 reveals biological data on the pure 6S diastereoisomer of compound 1, but does not disclose how this isomer was made.
Tri-O-t-butyl L-glutamyl-γ-D-glutamate, 4 can be purchased commercially as its N-benzyloxycarbonyl-protected precursor 10, which is stable, and it is conveniently deblocked to free amine 4 by catalytic hydrogenation shortly prior to use. However, although the two starting materials for dipeptide 4, N-Cbz-L-Glu, 11 and D-Glu 12 are not very expensive, the two t-butyl esters N-Cbz-Glu-α-O-t-Bu 13, obtained in only 33% yield from 11 in the one step preparation, and di-O-t-butyl-D-glutamate 14, are expensive, with the result that compound 10, as produced in Scheme 3, is very expensive.

Although there are higher yielding preparations of 13, they are multistep processes, which do not reduce the cost of the compound. Therefore, dipeptide 10 is very expensive, even on large scales. Thus, an improved synthesis of 10, or use of a less expensive form of the (R)-2-((S)-4-amino-4-carboxybutanamido)pentanedioic acid dipeptide, (hereafter referred to as either L-Glu-γ-D-Glu or L-Glutamyl-γ-D-Glutamic acid) in the coupling reaction, could have very beneficial effects on the production costs for compound 1.
As set out above, the prior art methods for addition of the dipeptide unit of compound 1 to the heterocyclic core (i.e. molecules 3 and 7) involve a coupling step and one or two deprotecting steps before isolation of the final product. This chemistry has several flaws, in addition to its high cost, which make it very poorly suited to commercially manufacturing a drug.
The reported chemistry was carried out on a very small scale, with no detailed analysis of the purity profile of the product 1. Examination of compound 1 under acidic conditions, with appropriate analytical techniques not previously described for compound 1, reveals it to have limited stability below pH 5 in aqueous solution. It has also been found that the simple monotrifluoroacetate salt of 1 has limited stability, both in the solid form and in trifluoroacetic acid (TFA) solution.
As removal of the t-butyl esters from 5, or its 2-depivaloylated equivalent, require strongly acidic conditions, this is very constraining on the actual set-up required for the deprotection. Trifluoroacetic acid can be stripped from the reaction mixture. However, the stability problems of 1 in TFA solution require that, as the scale is increased, the removal of the trifluoroacetic acid must be done at low temperature and relatively quickly. Without very specialized apparatus, this cannot easily be scaled up to any great extent. It is suggested that this chemistry would become totally impractical when producing API (Active Pharmaceutical Ingredient) in the 0.5 to 1 kg scale.
Furthermore, as this compound is used parenterally, the insoluble free acid 1 cannot be the drug product, and the above-referenced patent applications do not teach an efficient preparation and adequate purification of the sodium salt 2, which is the form in the final drug product. Due of the potent anti-cancer activity of 2, superior methods of synthesis of 2 have great value and utility.